This work was supported by the key projects of Chinese Academy of Sciences (KFZD-SW-205), strategic biological resources technology support system of Chinese Academy of Sciences (CZBZX-1).

Diese Studie wurde von der Ethikkommission des Instituts für Genetik und Entwicklungsbiologie genehmigt, und das Protokoll folgt den Richtlinien des Instituts für das Wohlergehen der Menschen. 1. Herstellung von EB-Virus <ol><li> Am Tag 1 auftauen B95-8 Zellen und der Kultur in 6 ml komplettem RPMI-1640-Medium, das mit 10% fötalem Rinderserum, 2 mM L-Glutamin und Antibiotika (100 ug / ml Streptomycin, 100 U / ml Penicillin) in einem ergänzten T25 Kulturflasche. Legen Sie die Kulturflasche in 5% CO 2 Inkubator bei 37 ° C. </li><li> Am Tag 2 werden die Zellmorphologie unter einem Mikroskop zu beobachten. Die Zellen sind in einem guten Zustand, wenn sie hell und rund sind. In 10 ml komplettem RPMI-1640-Medium. </li><li> Am Tag 4, 10 ml frisches vollständiges RPMI-1640-Medium, und dann in einen T75-Kulturkolben übertragen. </li><li> Am Tag 8 20 ml komplettem RPMI-1640-Medium. </li><li> Pflegen Sie die Zellen für 8 Tage ohne Zugabe von frischem Medium, das heißt, Hunger. </li><li> Am Tag 16, beobachten Zellenunter dem Mikroskop beobachtet. Sammle die Zellen in einem 15 ml-Zentrifugenröhrchen wurden 12 ml / Röhrchen. </li><li> für 1 h bei -80 ° C Cryo-bewahren, und dann bei 37 ° C schnell aufgetaut. 3-mal wiederholen, bis die Viruspartikel aus den Zellen freigesetzt werden. </li><li> Zentrifuge bei 240 × g für 10 min bei Raumtemperatur. </li><li> Filtern Sie die Überstand durch ein 0,22-um-Filter, aliquoten, 12 ml / Röhrchen, und bei -80 ° C. </li></ol> 2. Behandlung von Vollblut mit der roten Blutkörperchen Lysepuffer <ol><li> 0,5 mL Vollblut durch EDTA antikoaguliertem · K 2 in ein 15 ml Röhrchen. </li><li> Geben Sie 1 ml der roten Blutkörperchen Lysepuffer (gefiltert durch 0,22 um Filter und Auftauen auf Raumtemperatur vor dem Gebrauch) mit dem Vollblut und gut mischen. Legen Sie die Mischung bei Raumtemperatur für 4 min. Dann fügen Sie 5-7 ml 1x PBS schnell auf die Stoppreaktion. </li><li> Zentrifuge bei 240 × g für 5 min bei Raumtemperatur. Weiße Blutkörperchen werden am Boden des Röhrchens auszufällen. </li><li> Überstand verwerfen und waschen Sie die Fällung durch Zugabe von 7 ml 1640 Basismedium. Zentrifuge für 5 Minuten bei 240 xg bei Raumtemperatur. Zweimal wiederholen. </li><li> Resuspendieren des Zellpellets in 300 &amp; mgr; l Transformationsmedium (RPMI 1640 Medium, 20% fötales Rinderserum und 10 ug / ml Phytohämagglutinin (PHA)). </li></ol> 3. EBV-Infektion <ol><li> Hinzuzufügen 60 ul 20 ug / ml Cyclosporin A (CsA) zu der Zellsuspension. </li><li> Schnell ein Aliquot von EBV auftauen bei 37 ° C. Hinzufügen 240 uL EBV zu der Zellsuspension. </li><li> Gut mischen und legen Sie die Zellsuspension auf eine 96-Well-Platte, 200 &amp; mgr; l pro Vertiefung. Verdunstung, 200 ul 1x PBS zu jedem gut um der 96-Well-Platte zu vermeiden. Die Platte wird in einem 5% CO 2 Inkubator bei 37 ° C. </li></ol> 4. Erweiterung und Kryokonservierung von B-LCL <ol><li> Ändern Sie die Hälfte des Mediums einmal pro Woche im ersten Monat nach der EBV-Infektion. Nur eine dicke Layer von Zellbruchstücke sichtbar ist unter dem Mikroskop am Tag 7 (Abbildung 2). Nach einem Monat der EBV - Infektion sind lymphoblastoiden Zellcluster sichtbar unter einer dicken Schicht von Zellfragmente unter dem Mikroskop (Figur 2). </li><li> Ändern Sie die Hälfte des Mediums alle 3 Tage , bis die Zellhaufen deutlich sichtbar unter dem Mikroskop (Abbildung 2) sind. Typischerweise dauert dieser Vorgang 10 bis 20 Tage. </li><li> Nach dem Mischen pipettieren die Zellen und Transfer zum 24-Well-Platte. Die doppelte Volumen des Kulturmediums mit komplettem RPMI 1640, während es gelb wird. Dieses Verfahren hängt von der Rate des Zellwachstums. </li><li> Anschließend übertragen die Zellen zu T25 Kulturflasche. Die doppelte Volumen des Kulturmediums, wie es gelb wird, bis das Volumen des Mediums auf 10 erweitert - 15 ml. </li><li> Ändern Sie Medium 24 h vor dem Einfrieren. Zentrifuge Zellsuspension für 5 min bei 240 x g. Zählen von Zellen mit einem Zellzähler und gefrier bei einer Dichte von von 5 bis 10 x 10 6 / ml in frozen Stammlösung 90% FBS und 10% Dimethylsulfoxid (DMSO) enthält. Einfrieren bei einer Rate von 1 ° C pro min auf -80 ° C und übertragen dann direkt in flüssigem Stickstoff. </li></ol> 5. 3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyl-tetrazoliumbromid (MTT) Assay 17 <ol><li> Bereiten Sie eine B-LCL Einzelzellsuspension mit dem 1640-Medium, das 10% fötales Rinderserum enthält. Seed 3 x 10 4 Zellen pro Vertiefung auf 96-Well - Platte, 100 &amp; mgr; l pro Vertiefung. </li><li> Die Platte wird in 5% CO 2 Inkubator bei 37 ° C für 12 - 16 Uhr. </li><li> In MTT (5 mg / ml) zu den Vertiefungen, in denen Zellen wachsen und halten weg von Licht für 4 h bei 37 ° C. </li><li> Zentrifugenzellenplatte für 10 min bei 168 x g. </li><li> Überstand entfernen und mit 100 &amp; mgr; l DMSO pro Vertiefung. HINWEIS: Seien Sie vorsichtig. Berühren Sie nicht die nadelförmigen Farbstoffkristalle. </li><li> Schütteln Sie die Platte für 10 Minuten bei niedriger Geschwindigkeit vollständig um die Kristalle aufzulösen. </li><li> Measure Absorption bei 570 nm durchunter Verwendung eines Mikrotiterplatten-Spektrophotometer. </li></ol>

<ol>
	<li>Mohyuddin, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+instability+in+EBV-transformed+lymphoblastoid+cell+lines.">Genetic instability in EBV-transformed lymphoblastoid cell lines.</a> Biochim Biophys Acta. 1670 (1), 81-83 (2004).</li><li>Hu, V. W., Frank, B. C., Heine, S., Lee, N. 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L., Kunkel, H. 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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+method+for+the+rapid+establishment+of+Epstein-Barr+virus+immortalization+of+human+B+lymphocytes.">An efficient method for the rapid establishment of Epstein-Barr virus immortalization of human B lymphocytes.</a> Cell Prolif. 36 (4), 191-197 (2003).</li><li>Louie, L. G., King, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel-Approach+to+Establishing+Permanent+Lymphoblastoid+Cell-Lines+-+Epstein-Barr-Virus+Transformation+of+Cryopreserved+Lymphocytes.">A Novel-Approach to Establishing Permanent Lymphoblastoid Cell-Lines - Epstein-Barr-Virus Transformation of Cryopreserved Lymphocytes.</a> Am J Hum Genet. 48 (3), 637-638 (1991).</li><li>Tremblay, S., Khandjian, E. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+use+of+long-term+frozen+lymphocytes+for+the+establishment+of+lymphoblastoid+cell+lines.">Successful use of long-term frozen lymphocytes for the establishment of lymphoblastoid cell lines.</a> Clin Biochem. 31 (7), 555-556 (1998).</li><li>Reidy, J. A., Wheeler, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sample+age+and+Epstein-Barr+virus+transformation+of+cryopreserved+lymphocytes.">Sample age and Epstein-Barr virus transformation of cryopreserved lymphocytes.</a> In Vitro Cell Dev Biol. 28 (6), 383-384 (1992).</li><li>Amoli, M. M., Carthy, D., Platt, H., Ollier, W. E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EBV+immortalization+of+human+B+lymphocytes+separated+from+small+volumes+of+cryo-preserved+whole+blood.">EBV immortalization of human B lymphocytes separated from small volumes of cryo-preserved whole blood.</a> Int J Epidemiol. 37, 41-45 (2008).</li><li>Lacelle, C., Wang, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+lymphoblastoid+cell+lines+from+frozen+blood+of+extremely+old+individuals.">Establishing lymphoblastoid cell lines from frozen blood of extremely old individuals.</a> Mech Ageing Dev. 123 (10), 1415-1418 (2002).</li><li>Tohda, H., Oikawa, A., Kudo, T., Tachibana, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+greatly+simplified+method+of+establishing+B-lymphoblastoid+cell+lines.">A greatly simplified method of establishing B-lymphoblastoid cell lines.</a> Cancer Res. 38 (10), 3560-3562 (1978).</li><li>Ventura, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+Simple+Method+for+the+Epstein-Barr+Virus+Transformation+of+Lymphocytes+from+Members+of+Large+Families+of+Reunion+Island.">Use of a Simple Method for the Epstein-Barr Virus Transformation of Lymphocytes from Members of Large Families of Reunion Island.</a> Hum Hered. 38 (1), 36-43 (1988).</li><li>Chenevixtrench, G., Kerr, B., Walters, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphoblastoid+Cell-Lines+from+Frozen+Whole-Blood+-+a+Quick+and+Economical+Safeguard+for+Linkage+Analysis.">Lymphoblastoid Cell-Lines from Frozen Whole-Blood - a Quick and Economical Safeguard for Linkage Analysis.</a> Am J Hum Genet. 46 (1), 635-636 (1990).</li><li>Hansen, M. B., Nielsen, S. E., Berg, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Re-Examination+and+Further+Development+of+a+Precise+and+Rapid+Dye+Method+for+Measuring+Cell-Growth+Cell+Kill.">Re-Examination and Further Development of a Precise and Rapid Dye Method for Measuring Cell-Growth Cell Kill.</a> J Immunol Methods. 119 (2), 203-210 (1989).</li><li>Bogoslovsky, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preservation+and+enumeration+of+endothelial+progenitor+and+endothelial+cells+from+peripheral+blood+for+clinical+trials.">Preservation and enumeration of endothelial progenitor and endothelial cells from peripheral blood for clinical trials.</a> Biomark Med. 9 (7), 625-637 (2015).</li><li>Ambach, A., Bonnekoh, B., Gollnick, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Routine+flow+cytometric+immuno-staining+of+T-cell+perforin+is+preserved+using+diethylene+glycol+for+erythrocyte-lysis+but+lost+by+the+use+of+ammonium+chloride.">Routine flow cytometric immuno-staining of T-cell perforin is preserved using diethylene glycol for erythrocyte-lysis but lost by the use of ammonium chloride.</a> Exp Dermatol. 12 (6), 825-831 (2003).</li><li>Beck, J. C., Beiswanger, C. M., John, E. M., Satariano, E., West, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+transformation+of+cryopreserved+lymphocytes:+A+resource+for+epidemiological+studies.">Successful transformation of cryopreserved lymphocytes: A resource for epidemiological studies.</a> Cancer Epidemiol Biomarkers Prev. 10 (5), 551-554 (2001).</li><li>Tosato, G., Cohen, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+Epstein-Barr+Virus+(EBV)-immortalized+B+cell+lines.">Generation of Epstein-Barr Virus (EBV)-immortalized B cell lines.</a> Curr Protoc Immunol. , 22 (2007).</li></ol>

The authors declare that they have no competing financial interests.

Wir berichten über die Entwicklung eines neuartigen Verfahrens zum Lysieren von roten Blutkörperchen menschlichen B-Zellen aus Vollblut Immortalisierung und seine Lebensfähigkeit der Zellen von etablierten B-LCLs durch den MTT-Test bewertet. Die Ergebnisse zeigten, dass die Lebensfähigkeit der Zellen von B-LCLs durch die roten Blut Verfahren Zelllyse eingerichtet ist viel höher als die des Dichtegradienten Trennungsverfahren. Der große Vorteil der roten Blutkörperchen Verfahren der Lyse ist, dass es einfach ist, ...

Resting B cells could be transformed by the Epstein-Barr virus (EBV) in vitro into actively proliferating B lymphoblastoid cell lines (B-LCLs). B-LCLs are similar to germ cells in differentiation and development, and the somatic mutation rate of B-LCLs was only 0.3%1, with negligible genetic and phenotypic alteration. Therefore, B-LCLs, as surrogates for peripheral blood mononuclear cells (PBMCs), have substantially accelerated the progress of genomics, transcriptomics and proteomics study of EBV associated diseases2,3. Moreover, B-LCLs also have important application in screening monoclonal antibodies4,5. 
Several methods have been developed for the immortalization of B lymphocytes by EBV. The most major common procedures include the isolation of B lymphocytes and the use of B95-8 cell lines to produce infectious EBV. From isolation to transformation, these methods are divided into three strategies. Lymphocytes are separated from fresh blood by density gradient centrifugation and EBV transformation directly6-8, named density gradient separation method in this study. However, other techniques overcome the difficulty of shipping fresh blood and use a procedure for freezing purified lymphocytes and subsequent thawing and EBV infection9-11. Cryopreserved whole blood can also be used for the isolation of B lymphocytes and transformation12,13. In addition to density gradient centrifugation to separate B lymphocytes, some researchers use a magnetic cell sorter to select B lymphocytes12. However, magnetic separation is an expensive, complex and time consuming process. Although these methods are useful to immortalize B lymphocytes with a high success rate, the need to treat a large number of clinical samples and the relatively small volume of blood require a simpler method for the establishment of a permanent cell line.
Another less commonly used method is using whole blood as the source of nucleated cells for EBV infection and the establishment of B-LCLs. These methods simplify the technique by omitting the separation procedure. Only a small amount of whole blood, either fresh14 or frozen15,16, is sufficient to obtain the cell lines. Unfortunately, there is great variability in success from documents15,16 and several of our validation tests. Furthermore, transformed colonies are hardly recognized under a microscope because of the large contaminating impurities. Thus, a more reliable method for transformation is required.
Based on the above considerations, we have developed a novel method to establish B-LCLs without previous purification of the lymphocytes, named the red blood cell lysis method. This method is convenient, time saving and applicable to a large number of samples. As little as 0.5 mL of whole blood was enough to obtain B-LCLs that are easily obtainable from infants and the elderly without significantly harming their health.

<table><tbody><tr><td>Centrifuge</td><td>Techcomp</td><td>CT6T</td><td>Centrifugation</td></tr><tr><td>Automated Cell Counter&amp;nbsp;</td><td>Countstar</td><td>&amp;nbsp;IC 1000&amp;nbsp;</td><td>For cell counting&amp;nbsp;</td></tr><tr><td>Epoch Microplate Spectrophotometer</td><td>BioTek Instruments</td><td>SN263839</td><td>For measuring the absorbance</td></tr><tr><td>96 well cell culture cluster</td><td>Corning Incorporated</td><td>3599</td><td>Polystyrene plates</td></tr><tr><td>24 well cell culture cluster</td><td>Corning Incorporated</td><td>3524</td><td>Polystyrene plates</td></tr><tr><td>25 cm&lt;sup&gt;2 &lt;/sup&gt; cell culture flask</td><td>Nest</td><td>707001</td><td>Polystyrene&amp;nbsp;</td></tr><tr><td>FBS</td><td>Gibco</td><td>10270</td><td>Component of B-LCLs medium</td></tr><tr><td>RPMI Medium 1640</td><td>Gibco</td><td>31800-022</td><td>For B-LCLs medium</td></tr><tr><td>L-Glutamine&amp;nbsp;&amp;nbsp;</td><td>Amresco</td><td>0374</td><td>Component of B-LCLs medium</td></tr><tr><td>100x streptomycin penicillin solution BioRoYeeBRY-2309</td><td>BioRoYee</td><td>BRY-2309</td><td>Component of B-LCLs medium</td></tr><tr><td>Ficoll paque plus</td><td>GE&amp;nbsp; Healthcare</td><td>17-1440-03</td><td>For &lt;em&gt;in vitro&lt;/em&gt; isolation of lymphocyte</td></tr><tr><td>PHA-M</td><td>Sigma</td><td>L8902</td><td>For stimulating lymphocyte proliferation</td></tr><tr><td>Cyclosporin A</td><td>Cayman&amp;nbsp; Chemical</td><td>12088</td><td>For inhibiting the cytotoxicity effect of T cells</td></tr><tr><td>Red cell lysis buffer</td><td>Tiangen</td><td>RT122-01</td><td>For lysing red blood cell</td></tr><tr><td>MTT</td><td>Amresco</td><td>0793</td><td>For the detection of cell viability</td></tr><tr><td>DMSO</td><td>Sigma</td><td>D2650</td><td>For freezing cells</td></tr></tbody></table>

a simple red blood cell lysis method for the establishment of b lymphoblastoid cell lines

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new method with a powerful and simple strategy for the establishment of B-LCLs, the red blood cell lysis method. This method simplified the PBMC separation procedure with red blood cell removal, and used as little as 0.5 mL of whole blood for establishing EBV-immortalized cell lines, which can proliferate to large cell numbers in a relatively short amount time with a 100% success rate. The method is simple, reliable, time saving, and applicable to treating a large number of the clinical samples.

Während des Prozesses der Transformation werden die morphologischen Veränderungen der Zellen durch Lichtmikroskopie (Abbildung 2) visualisiert. Kleine Gruppen von Zellen waren deutlich sichtbar mit dem Dichtegradienten Trennverfahren nach 7 Tagen nach der Infektion, während nur eine dicke Schicht aus Zellfragmente aus der roten Blutkörperchen Lyseverfahren sichtbar sind. Allerdings sind Lymphoblastoidzelllinien Cluster sichtbar unter der dicken Schicht von Zellfragme...

Watch this Scientific Journal Video about A Simple Red Blood Cell Lysis Method for the Establishment of B Lymphoblastoid Cell Lines at JoVE.com

A Simple Red Blood Cell Lysis Method for the Establishment of B Lymphoblastoid Cell Lines

Eine einfache roten Blutkörperchen die Lyse Verfahren zum Aufbau B-LCLs mit hoher Immortalisierung Effizienz entwickelt, eine kleine Menge von Blut benötigt und die Zeit von der Initiierung der Gefrierkonservierung zu speichern.

A Simple Red Blood Cell Lysis Verfahren zur Etablierung von B Lymphoblastoidzelllinien

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new ...

a-simple-red-blood-cell-lysis-method-for-establishment-b

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

14.8K Aufrufe.  Chinese Academy of Sciences.  Eine einfache roten Blutkörperchen die Lyse Verfahren zum Aufbau B-LCLs mit hoher Immortalisierung Effizienz entwickelt, eine kleine Menge von Blut benötigt und die Zeit von der Initiierung der Gefrierkonservierung zu speichern. 

Serie: A Simple Red Blood Cell Lysis Method for the Establishment of B Lymphoblastoid Cell Lines

Recombinant Retroviral Production and Infection of B Cells

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control of a heterologous expression system to facilitate the analysis of the resulting phenotype. This approach can be used to express a gene that is not normally found in the organism, to express a mutant form of a gene product, or to over-express a dominant-negative form of the gene product. It is particularly useful in the study of the hematopoetic system, where transcriptional regulation is a major control mechanism in the development and differentiation of B cells 1, reviewed in 2-4.
Mouse genetics is a powerful tool for the study of human genes and diseases. A comparative analysis of the mouse and human genome reveals conservation of synteny in over 90% of the genome 5. Also, much of the technology used in mouse models is applicable to the study of human genes, for example, gene disruptions and allelic replacement 6. However, the creation of a transgenic mouse requires a great deal of resources of both a financial and technical nature. Several projects have begun to compile libraries of knock out mouse strains (KOMP, EUCOMM, NorCOMM) or mutagenesis induced strains (RIKEN), which require large-scale efforts and collaboration 7. Therefore, it is desirable to first study the phenotype of a desired gene in a cell culture model of primary cells before progressing to a mouse model. 
Retroviral DNA integrates into the host DNA, preferably within or near transcription units or CpG islands, resulting in stable and heritable expression of the packaged gene of interest while avoiding transcriptional silencing 8 9. The genes are then transcribed under the control of a high efficiency retroviral promoter, resulting in a high efficiency of transcription and protein production. Therefore, retroviral expression can be used with cells that are difficult to transfect, provided the cells are in an active state during mitosis. Because the structural genes of the virus are contained within the packaging cell line, the expression vectors used to clone the gene of interest contain no structural genes of the virus, which both eliminates the possibility of viral revertants and increases the safety of working with viral supernatants as no infectious virions are produced 10. 
Here we present a protocol for recombinant retroviral production and subsequent infection of splenic B cells. After isolation, the cultured splenic cells are stimulated with Th derived lymphokines and anti-CD40, which induces a burst of B cell proliferation and differentiation 11. This protocol is ideal for the study of events occurring late in B cell development and differentiation, as B cells are isolated from the spleen following initial hematopoetic events but prior to antigenic stimulation to induce plasmacytic differentiation.

An efficient system of structure and function analysis of a gene in an ex vivo culture of splenic B-lymphocytes is described. This method takes advantage of recombinant retroviral production in a helper free, ecotrophic packaging cell line. Stable, heritable expression of a gene of interest within primary lymphocytes is achieved leading to generation of surface antibodies on B cells undergoing class switch recombination.

Rekombinanten retroviralen Produktion und Infektion von B-Zellen

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control ...

Forschung

Immunologie und Infektion

Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid cell lines (LCL) in vitro. Since LCL are latently infected with EBV, they provide a model system to investigate EBV latency and virus-driven B cell proliferation and tumorigenesis1. LCL have been used to present antigens in a variety of immunologic assays2, 3. In addition, LCL can be used to generate human monoclonal antibodies4, 5 and provide a potentially unlimited source when access to primary biologic materials is limited6, 7.
A variety of methods have been described to generate LCL. Earlier methods have included the use of mitogens such as phytohemagglutinin, lipopolysaccharide8, and pokeweed mitogen9 to increase the efficiency of EBV-mediated immortalization. More recently, others have used immunosuppressive agents such as cyclosporin A to inhibit T cell-mediated killing of infected B cells7, 10-12.
The considerable length of time from EBV infection to establishment of cell lines drives the requirement for quicker and more reliable methods for EBV-driven B cell growth transformation. Using a combination of high titer EBV and an immunosuppressive agent, we are able to consistently infect, transform, and generate LCL from B cells in peripheral blood. This method uses a small amount of peripheral blood mononuclear cells that are infected in vitroclusters of cells can be demonstrated. The presence of CD23 with EBV in the presence of FK506, a T cell immunosuppressant. Traditionally, outgrowth of proliferating B cells is monitored by visualization of microscopic clusters of cells about a week after infection with EBV. Clumps of LCL can be seen by the naked eye after several weeks. We describe an assay to determine early if EBV-mediated growth transformation is successful even before microscopic clusters of cells can be demonstrated. The presence of CD23hiCD58+ cells observed as early as three days post-infection indicates a successful outcome.

We describe a method for generating transformed B cell lines using Epstein-Barr virus. We also illustrate a novel assay that can identify B cells destined to undergo transformation as early as three days after infection.

Gründung des Epstein-Barr Virus Growth-transformierten lymphoblastoiden Zelllinien

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid ...

A Chromatin Immunoprecipitation Assay to Identify Novel NFAT2 Target Genes in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant B cell clones and represents the most common leukemia in western countries. The majority of CLL patients show an indolent course of the disease as well as an anergic phenotype of their leukemia cells, referring to a B cell receptor unresponsive to external stimulation. We have recently shown that the transcription factor NFAT2 is a crucial regulator of anergy in CLL. A major challenge in the analysis of the role of a transcription factor in different diseases is the identification of its specific target genes. This is of&#160;great significance for the elucidation of pathogenetic mechanisms and potential therapeutic interventions. Chromatin immunoprecipitation (ChIP) is a classic technique to demonstrate protein-DNA interactions and can, therefore, be used to identify direct target genes of transcription factors in mammalian cells. Here, ChIP was used to identify LCK as a direct target gene of NFAT2 in human CLL cells. DNA and associated proteins are crosslinked using formaldehyde and subsequently sheared by sonication into DNA fragments of approximately 200-500 base pairs (bp). Cross-linked DNA fragments associated with NFAT2 are then selectively immunoprecipitated from cell debris using an &#945;NFAT2 antibody. After purification, associated DNA fragments are detected via quantitative real-time PCR (qRT-PCR). DNA sequences with evident enrichment represent regions of the genome which are targeted by NFAT2 in vivo. Appropriate shearing of the DNA and the selection of the required antibody are particularly crucial for the successful application of this method. This protocol is ideal for the demonstration of direct interactions of NFAT2 with target genes. Its major limitation is the difficulty to employ ChIP in large-scale assays analyzing the target genes of multiple transcription factors in intact organisms.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the western world. NFAT transcription factors are important regulators of development and activation in numerous cell types. Here, we present a protocol for the use of chromatin immunoprecipitation (ChIP) in human CLL cells to identify novel target genes of NFAT2.

Ein Chromatin Immunopräzipitation Assay, neuartige NFAT2 Zielgene in chronischer lymphatischer Leukämie zu identifizieren

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant B cell clones and represents the most common leukemia in western ...

Krebsforschung

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

Isolation und Aktivierung von murinen Lymphozyten

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration and differentiation, and cytokine production. All of these events are tightly linked to the energy status of the cell, and therefore studying the energy-producing pathways may give clues about the overall functionality of these cells. The extracellular flux analyzer is a commonly used device for evaluating the performance of glycolysis and mitochondrial respiration in many cell types. This system has been used to study immune cells in a few published reports, yet a comprehensive protocol optimized particularly for lymphocytes is lacking. Lymphocytes are fragile cells that survive poorly in ex vivo conditions. Oftentimes lymphocyte subsets are rare, and working with low cell numbers is inevitable. Thus, an experimental strategy that addresses these difficulties is required. Here, we provide a protocol that allows for rapid isolation of viable lymphocytes from lymphoid tissues, and for the analysis of their metabolic states in the extracellular flux analyzer. Furthermore, we provide results of experiments in which the metabolic activities of several lymphocyte subtypes at different cell densities were compared. These observations suggest that our protocol can be used to achieve consistent, well-standardized results even at low cell concentrations, and thus it may have broad applications in future studies focusing on the characterization of metabolic events in immune cells.

The study of metabolism is becoming increasingly relevant to immunological research. Here, we present an optimized method for measuring glycolysis and mitochondrial respiration in mouse splenocytes, and T and B lymphocytes.

Ein optimiertes Protokoll Glykolyse und die mitochondriale Atmung in Lymphozyten zu analysieren

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration ...

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or cellular processes. With the advancement of biotechnologies and the high translational potential of small molecules, a number of innovative approaches in drug discovery have evolved, which explains the resurgent interest in the use of HTS. The oncology field is currently the most active research area for drug screening, with no major breakthrough made for the identification of new immunomodulatory compounds targeting transplantation-related complications or autoimmune ailments. Here, we present a novel in vitro murine fluorescent-based lymphocyte assay easily adapted for the identification of new immunomodulatory compounds. This assay uses T or B cells derived from a transgenic mouse, in which the Nur77 promoter drives GFP expression upon T- or B-cell receptor stimulation. As the GFP intensity reflects the activation/transcriptional activity of the target cell, our assay defines a novel tool to study the effect of given compound(s) on cellular/biological responses. For instance, a primary screening was performed using 4,398 compounds in the absence of a &#34;target hypothesis&#34;, which led to the identification of 160 potential hits displaying immunomodulatory activities. Thus, the use of this assay is suitable for drug discovery programs exploring large chemical libraries prior to further in vitro/in vivo validation studies.

We present in the current study a novel fluorescence-based assay using lymphocytes derived from a transgenic mouse. This assay is suitable for high-throughput screening (HTS) of small molecules endowed with the capacity of either inhibiting or promoting lymphocyte activation.

Ein Fluoreszenz-basierte Lymphocyte Assay geeignet für Hochdurchsatz-Screening von kleinen Molekülen

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

Rapid Quantification of Mitogen-induced Blastogenesis in T Lymphocytes for Identifying Immunomodulatory Drugs

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., immunosuppressive or immunostimulatory) chemical compounds and biologics. One of the earliest steps during mitogenesis is cell enlargement or blastogenic transformation, whereupon the cell volume increases before division. It is usually detectable in the first several hours of T-lymphocyte stimulation. Here, we describe a rapid method to quantify blastogenesis in T lymphocytes isolated from mouse spleens and human peripheral blood mononuclear cells (PBMCs) using an automated cell counter. Various commonly used proliferation assays for the most part are laborious and only reflect the overall population effect rather than individual cellular effects within a population. In contrast, the presented automated cell counter assay provides rapid, direct, and precise measurements of cell diameters that can be used for assessing the effectiveness of various mitogens and immunomodulatory drugs in vitro.

T-lymphocyte mitogenesis is accompanied by blastogenic transformation, whereupon the cell volume enlarges before cell division. Here, we describe a method to quantify blastogenesis in T lymphocytes using an automated cell counter with the capability of measuring cell diameters.

Schnelle Quantifizierung von Mitogen-induzierte Blastogenese in T-Lymphozyten zur Identifizierung von immunmodulatorische Drogen

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., ...

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed feeder cells, purified NK or T cells with as much as 10 mL of blood, or a high-dose of IL-2. This study presents a new method with a powerful and simple strategy to establish NK and T cell lines by culturing the peripheral blood mononuclear cells (PBMC) with the addition of recombinant human IL-2 (rhIL-2), and uses as little as 2 mL of whole blood. The cells can proliferate quickly in two weeks and be maintained for more than 3 months. With this method, 7 NK or T cell lines have been established with a high success rate. This method is simple, reliable, and applicable to establishing cell lines from more cases of CAEBV or NK/T cell lymphoma.

A simple method for obtaining NK and T cell clones from CAEBV patients was developed with high efficiency, a small amount of peripheral blood, and a low-dose of IL-2.

Eine effiziente und einfache Methode, NK und T-Zell-Linien von Patienten mit chronischen aktiven Epstein - Barr-Virus-Infektion zu etablieren

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...

In Vitro Generation of Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors

Source: Chang, S., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/53211">In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System.</a> J. Vis. Exp. (2015)
This video demonstrates an in vitro assay to generate plasmacytoid dendritic cells (pDCs) from common lymphoid progenitor cells (CLPs). Upon co-culturing purified CLPs with &#947;-irradiated feeder cells in the presence of Fms-like tyrosine kinase-3 ligand (FL), the CLPs differentiate into non-adherent pDCs, the presence of which is confirmed using flow cytometry.

In vitro Erzeugung von plasmozytoiden dendritischen Zellen aus gemeinsamen lymphatischen Vorläuferzellen

Source: Chang, S., et al. In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System. J. ...

JoVE Encyclopedia of Experiments

Immunology

Immune Systems and Components

Cell Differentiation Techniques

Isolation and Purification of B Cells from Human Peripheral Blood

Source: Tzeng, S. J. <a href="https://appjove.unicartagenaproxy.elogim.com/v/54582">The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity.</a> J. Vis. Exp. (2016)
In this video, we demonstrate the isolation of B cells from human peripheral blood using biotinylated antibodies specific for non-B cells and streptavidin-conjugated magnetic microbeads.

Isolierung und Reinigung von B-Zellen aus menschlichem peripherem Blut

Source: Tzeng, S. J. The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of ...